The invention relates generally to the field of nucleic acid hybridization, and more particularly, to compositions and methods of nucleic acid amplification.
Agrawal, S. and Zamecnik, P. xe2x80x9cSite-specific functionalization of oligonucleotides for attaching two different fluorescent dye groupsxe2x80x9d, Nucleic Acids Research 18: 5419-5423 (1990). Andrus, A. xe2x80x9cChemical methods for 5xe2x80x2 non-isotopic labelling of PCR probes and primersxe2x80x9d
(1995) in PCR 2. A Practical Approach, Oxford University Press, Oxford, pp. 39-54.
Barany, F. xe2x80x9cThe Ligase Chain Reaction in a PCR Worldxe2x80x9d, PCR Methods and Applications 1:5-16 (1991).
Beaucage, S. and Iyer, R. xe2x80x9cAdvances in the synthesis of oligonucleotides by the phosphoramidite approachxe2x80x9d, Tetrahedron 48:2223-2311 (1992).
Bergot, B., Chakerian, V., Connell, C., Eadie, J., Fung, S., Hershey, N., Lee, L., Menchen, S. and Woo, S. xe2x80x9cSpectrally resolvable rhodamine dyes for nucleic acid sequence determinationxe2x80x9d, U.S. Pat. No. 5,366,860, issued Nov. 22, 1994.
Bevan etal, xe2x80x9cSequencing of PCR-amplified DNAxe2x80x9d, PCR Methods and Applications 1:222-228 (1992).
Blackburn, G. and Gait, M. Eds. xe2x80x9cDNA and RNA structurexe2x80x9d in Nucleic Acids in Chemistry and Biology, 2nd Edition, (1996) Oxford University Press, pp. 15-81.
Boffa, L., Carpaneto, E. and Allfrey, V. xe2x80x9cIsolation of active genes containing CAG repeats by DNA strand invasion by a peptide nucleic acidxe2x80x9d, PNAS (USA) 92:1901-05 (1995).
Bronstein, I. and Voyta, J., xe2x80x9cMethods of using chemiluminescent 1,2-dioxetanesxe2x80x9d U.S. Pat. No. 4,931,223, issued Jun. 5, 1990.
Bronstein, K., Fortin, J., Stanley, P., Stewart, G. and Kricka, L. xe2x80x9cChemiluminescent and bioluminescent reporter gene assaysxe2x80x9d, Anal. Biochemistry 219:169-81 (1994).
Cardullo, R., Agrawal, S., Flores, C., Zamecnik, P. and Wolf, D. xe2x80x9cDetection of nucleic acid hybridization by non-radiative fluorescence resonance energy transferxe2x80x9d, Proc. Natl. Acad. Sci. 85:8790-8794 (1988).
Caruthers, M. and Beaucage, S., xe2x80x9cPhosphoramidite compounds and processesxe2x80x9d U.S. Pat. No. No. 4,415,732, issued Nov. 15, 1983.
Clegg, R. xe2x80x9cFluorescence resonance energy transfer and nucleic acidsxe2x80x9d, Meth. Enzymol. 211:353-388 (1992).
Dueholm, K., Egholm, M., Behrens, C., Christensen, L., Hansen, H., Vulpius, T., Petersen, K., Berg, R., Nielsen, P. and Buchardt, O. xe2x80x9cSynthesis of peptide nucleic acid monomers containing the four natural nucleobases: thymine, cytosine, adenine, and guanine and their oligomerizationxe2x80x9d, J. Org. Chem. 59:5767-73 (1994).
Egholm, M., Buchardt, O., Christensen, L., Behrens, C., Freier, S., Driver, D., Berg, R. and Kim, S. xe2x80x9cPNA hybridizes to complementary oligonucleotides obeying the Watson-Crick hydrogen bonding rulesxe2x80x9d, Nature 365:566-68 (1993).
Egholm, M., Christensen, L., Dueholm, K., Buchardt, O., Coull, J. and Nielsen, P. xe2x80x9cEfficient pH-independent sequence-specific DNA binding by pseudoisocytosine-containing bis-PNAxe2x80x9d, Nucleic Acids Res. 23:217-22 (1995).
Englisch, U. and Gauss, D. xe2x80x9cChemically modified oligonucleotides as probes and inhibitorsxe2x80x9d, Angew. Chem. Int. Ed. Engl. 30:613-29 (1991).
Froehler, B., Wagner, R., Matteucci, M., Jones, R., Gutierrez, A. and Pudlo, J. xe2x80x9cEnhanced triple-helix and double-helix formation with oligodeoxyribonucleotides containing modified pyrimidinesxe2x80x9d U.S. Pat. No. 5,645,985, issued Jul. 8, 1997.
Froehler, B. and Matteucci, M. xe2x80x9cEnhanced triple-helix and double-helix formation with oligomers containing modified purinesxe2x80x9d, U.S. Pat. No. 5,594,121, issued Jan. 14, 1997.
Gelfand, D., Holland, P., Saiki, R., and Watson, R. xe2x80x9cHomogeneous assay system using the nuclease activity of a nucleic acid polymerasexe2x80x9d, U.S. Pat. No. 5,210,015, issued May 9, 1993.
Gong, B. and Yan, Y. xe2x80x9cNew DNA minor-groove binding molecules with high sequence-selectivities and binding affinitiesxe2x80x9d, Biochem. and Biophys. Res. Comm. 240:557-60 (1997).
Hermanson, G. in Bioconjugate Techniques (1996) Academic Press, San Diego, pp. 40-55, 643-671.
Higuchi, R., Fockler, C., Dollinger, G. and Watson, R. xe2x80x9cKinetic PCR: Real time monitoring of DNA amplification reactionsxe2x80x9d, Biotechnology 11:1026-30 (1993).
Higuchi, R., Dollinger, G., Walsh, P., and Griffith, R. xe2x80x9cSimultaneous amplification and detection of specific DNA sequencesxe2x80x9d, Biotechnology 10:413-17 (1992).
Holland, P., Abramson, R., Watson, R. and Gelfand, D. xe2x80x9cDetection of specific polymerase chain reaction product by utilizing the 5xe2x80x2 to 3xe2x80x2 exonuclease activity of Thermus aquaticus DNA polymerasexe2x80x9d, Proc. Natl. Acad. Sci. 88:7276-80 (1991).
Ju, J., Kheterpal, I., Scherer, J., Ruan, C., Fuller, C., Glazer, A. and Mathies, R. xe2x80x9cDesign and Synthesis of fluorescence energy transfer dye-labeled primers and their application for DNA sequencing and analysisxe2x80x9d, Analytical Biochemistry 231:131-140 (1995).
Koch, T., Hansen, H., Andersen, P., Larsen, T., Batz, H., Otteson, K. and Ørum, H. xe2x80x9cImprovements in automated PNA synthesis using BOC/Z monomersxe2x80x9d, J. Peptide Res. 49:80-88 (1997).
Kricka, L. in Nonisotopic DNA Probe Techniques (1992), Academic Press, San Diego, pp. 3-25 28.
Kubista, M. and Svanvik, N. xe2x80x9cProbe for analysis of nucleic acidsxe2x80x9d, WO 97/45539, Intl. Publ. Date Dec. 4, 1997.
Kutyavin, I., Lukhtanov, E., Gamper, H. and Meyer, R. xe2x80x9cCovalently linked oligonucleotide minor groove binder conjugatesxe2x80x9d, WO 96/32496, Intl. Publ. Date Oct. 17, 1996.
Kutyavin, I., Rhinehart, R., Lukhtanov, E., Gorn, V., Meyer, R. and Gamper, H. xe2x80x9cOligonucleotides containing 2-aminoadenine and 2-thiothymine act as selectively binding complementary agentsxe2x80x9d, Biochemistry 35:11170-11176 (1996).
Lawyer, F., Stoffel, S., Saiki, R., Myambo, K., Drummond, R. and Gelfand, D. xe2x80x9cIsolation, characterization, and expression in Escherichia coil of the DNA polymerase gene from the extreme thermophile, Thermus aquaticusxe2x80x9d, J. Biol. Chem. 264:6427-37 (1989).
Livak, K., Flood, S., Marmaro, J., Giusti, W. and Deetz, K. xe2x80x9cOligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridizationxe2x80x9d, PCR Methods and Applications 4:357-362 (1995).
Livak, K., Flood, S., Marmaro, J. and Mullah, K. xe2x80x9cSelf-quenching fluorescence probexe2x80x9d, U.S.
Pat. No. 5,723,591, issued Mar. 3, 1998.
Livak, K., Flood, S. and Marmaro, J. xe2x80x9cMethod for Detecting Nucleic Acid Amplification Using Self-Quenching Fluorescence Probexe2x80x9d, U.S. Pat. No. 5,538,848, issued Jul. 23, 1996.
Livak, K., Marmaro, J., and Todd, J. xe2x80x9cTowards fully automated genome-wide polymorphism screeningxe2x80x9d, Nature Genetics 9:341-42 (1995).
Lukhtanov, E., Kutyavin, I., Gamper, H. and Meyer, R. xe2x80x9cOligodeoxyribonucleotides with conjugated dihydropyrroloindole oligopeptides: Preparation and hybridization propertiesxe2x80x9d, Bioconjugate Chem. 6:418-26 (1995).
Lyman, S., Aster, R., Visentin, G. and Newman, P. xe2x80x9cPolymorphism of human platelet membrane glycoprotein IIb associated with Baka/Bakb alloantigen systemxe2x80x9d, Blood 75:2343-2348 (1990).
McPherson, M. J., Quirke, P., and Taylor, G. R. in PCR 2: A Practical Approach (1995) Oxford University Press, Oxford.
Menchen, S., Lee, L., Connell, C., Hershey, N., Chakerian, V., Woo, S. and Fung, S. xe2x80x9c4,7-Dichlorofluorescein dyes as molecular probesxe2x80x9d, U.S. patent 5,188,934, issued Feb. 23, 1993.
Meyer, R. xe2x80x9cIncorporation of modified bases in oligonucleotidesxe2x80x9d in Protocols for Oligonucleotide Conjugates, Ed. S. Agrawal (1 994) Humana Press, Totowa, N.J., pp. 73-92.
Mullah, B. and Andrus, A. xe2x80x9cAutomated synthesis of double dye-labeled oligonucleotides using tetramethylrhodamine (TAMRA) solid supportsxe2x80x9d, Tetrahedron Letters 38: 5751-5754 (1997).
Mullah, B. and Andrus, A. xe2x80x9cSolid support reagents for the direct synthesis of 3xe2x80x2-labeled polynucleotidesxe2x80x9d, U.S. Pat. No. 5,736,626, issued Apr. 7, 1998.
Mullah, B., Livak, K., Andrus, A. and Kenney, P. xe2x80x9cEfficient synthesis of double dye-labeled oligodeoxyribonucleotide probes and their application in a real time PCR assayxe2x80x9d, Nucl. Acids Res. 26:1026-1031 (1998).
Nielsen, P. and Christensen, L. xe2x80x9cStrand displacement binding of duplex-forming homopurine PNA to a homopyrimidine duplex DNA targetxe2x80x9d, Jour. Amer. Chem. Soc. 118:2287-88 (1996).
Nielsen, P., Egholm, M., Berg, R. and Buchardt, O. xe2x80x9cSequence-selective recognition of DNA by strand displacement with a thymidine-substituted polyamidexe2x80x9d, Science 254:1497-1500 (1991).
Pemble, S., Schroeder, K., Spencer, S., Meyer, D., Hallier, E., Bolt, H., Ketterer, B. and Taylor, J. B. xe2x80x9cHuman glutathion S-transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphismxe2x80x9d Biochem J. 300:271-276 (1994).
Rumney, S. and Kool, E. xe2x80x9cStructural optimization of non-nucleotide loop replacements for duplex and triplex DNAsxe2x80x9d J. Amer. Chem. Soc. 117:5636-46 (1995).
Theisen, P., McCollum, C. and Andrus, A. xe2x80x9cFluorescent dye phosphoramidite labelling of oligonucleotidesxe2x80x9d, in Nucleic Acid Symposium Series No. 27, Oxford University Press, Oxford, pp. 99-100 (1992).
Van der Laan, A., Brill, R., Kuimelis, R., Kuyl-Yeheskiely, E., van Boom, J., Andrus, A. and Vinayak, R. xe2x80x9cA convenient automated solid-phase synthesis of PNA-(5xe2x80x2)-DNA-(3xe2x80x2)-PNA chimeraxe2x80x9d, Tetrahedron Lett. 38:2249-52 (1997).
Vinayak, R., van der Laan, A., Brill, R., Otteson, K., Andrus, A., Kuyl-Yeheskiely, E. and van Boom, J. xe2x80x9cAutomated chemical synthesis of PNA-DNA chimera on a nucleic synthesizerxe2x80x9d, Nucleosides and Nucleotides 16:1653-56 (1997).
Walker, G. et al, xe2x80x9cStrand displacement amplificationxe2x80x94an isothermal, in vitro DNA amplification techniquexe2x80x9d, Nucleic Acids Research 20: 1691-1696 (1992).
Walker, G., Little, M., Nadeau, J. and Shank, D., xe2x80x9cIsothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase systemxe2x80x9d, Proc. Natl. Acad. Sci. 89:392-96 (1992).
Wittung, P., Nielsen, P. and Norden, B. xe2x80x9cExtended DNA-recognition repertoire of peptide nucleic acid (PNA): PNA-dsDNA triplex formation with cytosine-rich homopyrimidine PNAxe2x80x9d, Biochemistry 36:7973-79 (1997).
Woo, S., Menchen, S. and Fung, S. xe2x80x9cRhodamine phosphoramidite compoundsxe2x80x9d, U.S. Pat. No. 5,231,191, issued Jul. 27, 1993.
Woo, S. and Fung, S. xe2x80x9cSolid support reagents for the synthesis of 3xe2x80x2-nitrogen containing polynucleotidesxe2x80x9d, U.S. Pat. No. 5,552,471, issued Sep. 3, 1996.
Nucleic acid hybridization assays comprise an important class of techniques in modern biology, with diverse applications in diagnosis of inherited disease, human identification, identification of microorganisms, paternity testing, virology, and DNA sequencing. Primer extension reactions are key components of many nucleic acid hybridization assays and amplification methods. The polymerase chain reaction (PCR) amplification method has enabled advances in cloning, analysis of genetic expression, DNA sequencing, genetic mapping, drug discovery, and the like (Gilliland, 1990; Bevan, 1992; Green, 1991; McPherson, 1995).
The specificity and affinity of two or more strands of nucleic acid and nucleic acid analog molecules hybridizing by Watson/Crick and non-Watson/Crick base pairing are key parameters of efficient nucleic acid hybridization assays. Covalently attached labels, or moieties, which improve or stabilize hybridization are desirable.
Another important aspect of nucleic acid hybridization assays is the method used to facilitate detection of the hybridization event. Fluorescent methods have many advantages over radioisotopes where either the target polynucleotide or the probe or primer can be easily and safely labelled with fluorescent dyes. Methods to lower the costs of labelled probes and primers, or their functional equivalents, are highly desirable. There remains a need for further improvements in the specificity, affinity, and detection of nucleic acid hybridization assays.
The present invention is directed towards novel binary probe and clamp compositions which are useful in nucleic acid hybridization assays, and methods of using such compositions.
In a first aspect, the invention comprises a binary probe and clamp composition in which the probe comprises a target-specific portion and a clamp-specific portion. The target-specific portion is capable of sequence-specific binding to a target polynucleotide sequence. A clamp comprises a probe-specific portion and a label. The clamp-specific portion of the probe and the probe-specific portion of the clamp form a duplex structure in the binary probe and clamp composition. Alternatively, the clamp-specific portion of the probe and two probe-specific portions of the clamp form a triplex structure.
During a nucleic acid hybridization assay, the target polynucleotide sequence may be detected and quantitated in the sample by detection of the label on the binary composition. The clamp-specific portion of the probe is bound to the probe-specific portion of the clamp during the detection phase of the assay.
The clamp may be an oligonucleotide or a nucleic acid analog. The nucleic acid analog may be comprised of modifications to the internucleotide linkage, the sugar, or nucleobase moieties. A preferred clamp is 2-aminoethylglycine, PNA, (Nielsen, 1991) with the structure 
where B is a nucleobase or nucleobase analog.
The probe may be an oligonucleotide or a nucleic acid analog containing nucleobase analogs, sugar analogs, and/or internucleotide analogs.
In a second aspect, the probe or clamp in the binary composition has one or more labels. The probe label can be attached at sites including but not limited to a 5xe2x80x2 terminus, a 3xe2x80x2 terminus, a nucleobase, an internucleotide linkage, or a sugar. The clamp label can be attached at sites including a terminus, a nucleobase, an internucleotide linkage, a sugar, an amino group, a sulfide group, and a carboxyl group. The labels may include hybridization-stabilizing moieties, fluorescent dyes, fluorescence quenchers, chemiluminescent dyes, amino acids, and affinity ligands.
In a third aspect, a method is provided for detecting a target polynucleotide sequence with the binary probe and clamp composition. In the method, a target-specific portion of the probe hybridizes to a target polynucleotide sequence, and a labelled clamp hybridizes to the probe. The steps of (i) hybridizing the target-specific portion of the probe with the target polynucleotide sequence; (ii) hybridizing the probe-specific portion of the clamp with the clamp-specific portion of the probe; and (iii) detecting the binary probe are conducted to detect a target polynucleotide.
In a fourth aspect, a method is provided for detecting polymerase chain reaction products with a binary probe and clamp composition. In the method, the probe includes a fluorescent dye or quencher, and the clamp includes a fluorescent dye or quencher, such that the binary composition comprises one fluorescent dye and one quencher. The binary composition is largely self-quenching when the probe is hybridized to the clamp. The method is conducted via the steps of (i) hybridizing a target-specific portion of a probe with a target polynucleotide sequence; (ii) hybridizing a probe-specific portion of the clamp with a clamp-specific portion of the probe; (iii) amplifying the target with a DNA polymerase (Lawyer, 1989) with 5xe2x80x2 to 3xe2x80x2 exonuclease activity, PCR primers, and nucleoside 5xe2x80x2 triphosphates, (iv) cleaving the probe by the exonuclease activity of the polymerase, and (v) detecting the fluorescent dye. It is an object of the present invention to detect the labels by monitoring the emitted fluorescence in real-time or at the end-point of target amplification.
In a fifth aspect, a method is provided for labelling polymerase chain reaction products with a binary primer and labelled clamp composition. In the method, a primer has a target-specific portion, capable of sequence-specific binding to a target polynucleotide sequence, and a clamp-specific portion. The clamp includes a label and a primer-specific portion capable of sequence-specific binding to the clamp-specific portion of the primer. The label may be a detectable fluorescent dye. The clamp-specific portion of the primer and the primer-specific portion of the clamp form a duplex structure in the binary primer and clamp composition. Alternatively, the clamp-specific portion of the primer and two primer-specific base-pairing portions of the clamp form a triplex structure in the binary primer and clamp composition. A target polynucleotide is amplified with a DNA polymerase, one or more binary primer and clamp compositions, one or more opposing strand primers, and nucleoside 5xe2x80x2 triphosphates, wherein one or more labelled polymerase chain reaction products result.
In a sixth aspect, a method is provided for detecting polymerase chain reaction products where a target polynucleotide is amplified with a primer comprising a target-specific portion at a 3xe2x80x2 end and a homopyrimidine sequence portion at a 5xe2x80x2 end. A clamp comprising the same homopyrimidine sequence as the 5xe2x80x2 end of the target and one or more labels is present in the PCR mixture. The homopyrimidine sequence of the primer is amplified and forms a terminal part of the PCR product. The clamp forms a duplex or triplex structure by hybridization with the PCR product and the label is detected. The label can be a fluorescent dye.